Stromal cells suppress CD8+ T cell proliferation via a contact independent mechanism.

CAF exosomes alter TCR signaling, transcriptional regulation, and metabolic profiles in CD8+ T cells. A, Differentially expressed proteins were analyzed using volcano plots for comparisons between unstimulated murine CD8 T cells and T cells stimulated with anti-CD3/CD28 antibodies, after 48 hours, with GO Biological Process (GOBP) analysis shown in B. C, Differentially expressed proteins were analyzed using volcano plots for comparisons between murine CD8 T cells stimulated with anti-CD3/CD28 antibodies for 4 hours, after 24 hours in the presence of cancer cell or CAF exosomes, with GOBP analysis shown in D. E, Heatmap showing differentially expressed phosphorylated proteins in CD8 T cells exposed to cancer cells or CAF exosomes for 24 hours, followed by stimulated with anti-CD3/CD28 antibodies for 4 hours. F, Western blot of phosphorylated (p)ZAP-70 (Y352), total ZAP-70, pSLP-76 (S376), total SLP-76, pPLC-γ (Y783), total PLC-γ in unstimulated (-) CD8 T cells, and CD8 T cells stimulated with cancer cells or CAF exosomes (Exo.) and anti-CD3/CD28 antibodies for 15 minutes or 30 minutes.

PD-L1/PD-1 blockade fails to rescue CD8+ T cell proliferation in the presence of CAF exosomes.

Density gradient ultracentrifugation-based isolation of serum reveals fractions characterized by exosomal markers.

Effective anti-tumor immunity depends on cytotoxic T cells entering tumors, surviving there, and maintaining effector function. However, many breast cancers, particularly hormone receptor-positive subtypes, contain relatively few infiltrating T cells. This pattern reflects a hierarchy of processes, beginning with the generation of cancer-specific T cells. Once primed in lymph nodes, T cells exit nodes and enter tumors through blood vessels. This process is controlled by endothelial adhesion molecule expression and vessel integrity. Furthermore, chemokine gradients control whether T cells infiltrate cancer cell nests or are retained in the tumor stroma. Finally, the extracellular matrix adds physical and biochemical constraints through fiber alignment, cross-linking, and matricellular proteins that can limit T-cell extravasation and intratumoral positioning. These barriers are interdependent, as extracellular matrix stiffness compresses blood vessels, disrupting adhesion molecule expression and chemokine presentation. Emerging preclinical and early clinical strategies demonstrate that vascular normalization, chemokine reprogramming, and selective extracellular matrix remodeling can improve T-cell access and sensitize tumors to checkpoint blockade. Durable clinical benefit will likely require biomarker-guided combination approaches that both enable T-cell entry and preserve their function within the suppressive tumor microenvironment.

Cancer-associated fibroblasts (CAF), a major component of the breast tumor microenvironment, drive immune evasion in various cancers by promoting T-cell exclusion and dampening T-cell activation. Previous studies have implicated CAF-derived soluble factors in mediating these immunosuppressive effects. In this study, we investigated whether exosomes secreted by CAFs could suppress T-cell activity. Inhibition of global exosome secretion in breast tumor-bearing mice significantly reduced tumor growth and increased tumor-infiltrating T cells with lower exhaustion marker expression. Conversely, administration of CAF-derived exosomes into tumors produced the opposite effects. Moreover, CAF exosomes associated with T cells in vivo and impaired T-cell activation and cytotoxic potential in ex vivo assays. Proteomic and biochemical analyses of T cells exposed to CAF exosomes revealed dampened early T-cell receptor signaling. Mass spectrometry identified an extracellular matrix (ECM) signature on CAF exosomes. Depleting type I and type V collagens from CAF exosomes restored T-cell proliferation, whereas overexpression of collagen in cancer cells led to its incorporation into exosomes, which suppressed T-cell activation. These findings suggest that a signaling bridge between CAF exosomes and T cells, mediated by collagen, promotes T-cell dysfunction, contributing to immune evasion in breast cancer.Significance: Our data provide the first evidence that ECM proteins associate with mouse and human breast CAF-derived exosomes and directly impair T-cell activation and cytotoxicity. These findings suggest that signaling between collagen-rich CAF exosomes and T cells contribute to local and systemic T-cell dysfunction.

Black women in the United States experience disproportionately high breast cancer mortality and have high rates of comorbid hypertension; however, the associations of hypertension and antihypertensive medication use with breast cancer survival are unclear. We examined these associations among 2474 Black Women’s Health Study participants with invasive breast cancer. Hypertension and antihypertensive medication use were assessed biennially, and breast cancer diagnoses were confirmed through medical records and cancer registries. We used Cox proportional hazards models, adjusted for clinical and lifestyle factors and cancer treatment, to estimate hazard ratios (HR) for breast cancer-specific death. In the full study population, the HR for untreated hypertension compared to no hypertension was 1.17 (95

BACKGROUND:Lymphatic muscle cells (LMCs) are indispensable for lymphatic vessel contraction, and their aberrant recruitment or absence is associated with both primary and secondary lymphedema. Despite their critical role in lymphatic vessel function, the cellular and molecular bases that confer the unique contractile properties to LMCs are largely undefined, limiting the development of therapeutic interventions that precisely target LMCs. METHODS:We used single-cell RNA sequencing, genetic lineage tracing, whole mount immunostaining, and intravital imaging to investigate the basis for the hybrid cardiomyocyte and blood vascular smooth muscle cell (SMC) characteristics that have been described for LMCs. RESULTS:Using single-cell RNA sequencing, the transcriptomes of LMCs and venous SMCs exhibited more similarities than differences, with both cell types exhibiting enrichment in overlapping molecular markers. Notably, LMCs and venous SMCs were both markedly distinct from that of arteriole SMCs. Functionally, both lymphatic vessels and blood vessels in the murine hind limb displayed pulsatile contractility, and their functions were regulated by gabapentin and nifedipine, which target the activity of voltage-gated calcium channels. Although LMCs express genes that overlap with the venous SMC transcriptome, lineage tracing demonstrates that LMCs do not originate from Myh11 (myosin heavy chain 11) lineage-derived SMCs, Nkx2.5 (NK2 homeobox 5) cardiomyocyte progenitors, or Wnt1 (Wnt family member 1) neural crest progenitors. Instead, most LMCs and SMCs in the hind limb and inguinal-axillary region originate from WT1+ (Wilms tumor gene 1) mesodermal progenitors from the lateral plate mesoderm. LMCs derived from WT1+ progenitors were critical for the maintenance of lymphatic vessel contractility. CONCLUSIONS:Overall, our findings suggest that venous SMCs and LMCs derive from a related mesodermal progenitor and acquire a similar gene expression program that facilitates their contractile properties.

Lymphatic muscle cells (LMCs) are indispensable for lymphatic vessel contraction and their aberrant recruitment or absence is associated with both primary and secondary lymphedema. Despite their critical role in lymphatic vessel function, the transcriptomic and developmental basis that confer the unique contractile properties to LMCs are largely undefined. In this study, we employed single-cell RNA sequencing (scRNAseq), lineage tracing and in vivo imaging to investigate the basis for the hybrid cardiomyocyte and blood vascular smooth muscle cell (SMC) characteristics that have been described for LMCs. Using scRNAseq, the transcriptomes of LMC and venous SMCs from the murine hindlimb exhibited more similarities than differences, although both were markedly distinct from that of arteriole SMCs in the same tissue. Functionally, both lymphatic vessels and blood vessels in the murine hindlimb displayed pulsatile contractility. However, despite expressing genes that overlap with the venous SMC transcriptome, through lineage tracing we show that LMCs do not originate from Myh11+ SMC progenitors. Previous studies have shown that LMCs express cardiac-related genes, whereas in our study we found that arteriole SMCs, but not LMCs, expressed cardiac-related genes. Through lineage tracing, we demonstrate that a subpopulation of LMCs and SMCs originate from WT1+ mesodermal progenitors, which are known to give rise to SMCs. LMCs, however, do not derive from Nkx2.5+ cardiomyocyte progenitors. Overall, our findings suggest that venous SMCs and LMCs and may derive from a related mesodermal progenitor and adopt a similar gene expression program that enable their contractile properties.

Preclinical models that display spontaneous metastasis are necessary to improve the therapeutic options for hormone receptor-positive breast cancers. Within this study, detailed cellular and molecular characterization was conducted on MCa-P1362, a newly established mouse model of metastatic breast cancer that is syngeneic in BALB/c mice. MCa-P1362 cancer cells express estrogen receptor, progesterone receptor, and the human epidermal growth factor receptor 2. MCa-P1362 cancer cells proliferate in vitro and in vivo in response to estrogen, yet do not depend on steroid hormones for growth and tumor progression. Analysis of MCa-P1362 tumor explants revealed the tumors contained a mixture of cancer cells and mesenchymal stromal cells. Through transcriptomic and functional analyses of both cancer and stromal cells, stem cells were detected within both populations. Functional studies demonstrated that MCa-P1362 cancer stem cells drove tumor initiation, whereas stromal cells from these tumors contributed to drug resistance. MCa-P1362 may serve as a useful preclinical model to investigate the cellular and molecular basis of breast tumor progression and therapeutic resistance.

Cancer dissemination to lymph nodes (LN) is associated with a worse prognosis, increased incidence of distant metastases and reduced response to therapy. The LN microenvironment puts selective pressure on cancer cells, creating cells that can survive in LN as well as providing survival advantages for distant metastatic spread. Additionally, the presence of cancer cells leads to an immunosuppressive LN microenvironment, favoring the evasion of anti-cancer immune surveillance. However, recent studies have also characterized previously unrecognized roles for tumor-draining lymph nodes (TDLNs) in cancer immunotherapy response, including acting as a reservoir for pre-exhausted CD8+ T cells and stem-like CD8+ T cells. In this review, we will discuss the spread of cancer cells through the lymphatic system, the roles of TDLNs in metastasis and anti-cancer immune responses, and the therapeutic opportunities and challenges in targeting LN metastasis.

Lymphatic muscle cells (LMCs) are indispensable for proper functioning of the lymphatic system, as they provide the driving force for lymph transport. Recent studies have advanced our understanding of the molecular mechanisms that regulate LMCs, which control rhythmic contraction and vessel tone of lymphatic vessels-traits also found in cardiac and vascular smooth muscle. In this review, we discuss the molecular pathways that orchestrate LMC-mediated contractility and summarize current knowledge about their developmental origin, which may shed light on the distinct contractile characteristics of LMCs. Further, we highlight the growing evidence implicating LMC dysregulation in the pathogenesis of lymphedema and other diseases related to lymphatic vessel dysfunction. Given the limited number and efficacy of existing therapies to treat lymphedema, LMCs present a promising focus for identifying novel therapeutic targets aimed at improving lymphatic vessel contractility. Here, we discuss LMCs in health and disease, as well as therapeutic strategies aimed at targeting them to improve lymphatic vessel function.

Early in solid tumor development, antigens are presented in tumor-draining lymph nodes (tdLNs), a process that is necessary to set up immune surveillance. Recent evidence indicates that tdLNs fuel systemic tumor-specific T cell responses which may halt cancer progression and facilitate future responses to immunotherapy. These protective responses, however, are subject to progressive dysfunction exacerbated by lymph node (LN) metastasis. We discuss emerging preclinical and clinical literature indicating that the tdLN is a crucial reservoir for systemic immunity that can potentiate immune surveillance. We also discuss the impact of LN metastasis and argue that a better understanding of the relationship between LN metastasis and systemic immunity will be necessary to direct regional disease management in the era of immunotherapy.

Tumor-draining lymph nodes (TDLNs) are important for tumor antigen–specific T cell generation and effective anticancer immune responses. However, TDLNs are often the primary site of metastasis, causing immune suppression and worse outcomes. Through cross-species single-cell RNA-Seq analysis, we identified features defining cancer cell heterogeneity, plasticity, and immune evasion during breast cancer progression and lymph node metastasis (LNM). A subset of cancer cells in the lymph nodes exhibited elevated MHC class II (MHC-II) gene expression in both mice and humans. MHC-II+ cancer cells lacked costimulatory molecule expression, leading to regulatory T cell (Treg) expansion and fewer CD4+ effector T cells in TDLNs. Genetic knockout of MHC-II reduced LNM and Treg expansion, while overexpression of the MHC-II transactivator, Ciita, worsened LNM and caused excessive Treg expansion. These findings demonstrate that cancer cell MHC-II expression promotes metastasis and immune evasion in TDLNs.

PDF file - 1.8MB, Effect of homozygous or haploid Bmx deletion on tumor growth in female mice in the chemical skin carcinogenesis model

PDF file - 196K, Normal postnatal development of retinal vasculature in Bmx deficient mice

Lymphatic muscle cells (LMCs) within the wall of collecting lymphatic vessels exhibit tonic and autonomous phasic contractions, which drive active lymph transport to maintain tissue-fluid homeostasis and support immune surveillance. Damage to LMCs disrupts lymphatic function and is related to various diseases. Despite their importance, knowledge of the transcriptional signatures in LMCs and how they relate to lymphatic function in normal and disease contexts is largely missing. We have generated a comprehensive transcriptional single-cell atlas-including LMCs-of collecting lymphatic vessels in mouse dermis at various ages. We identified genes that distinguish LMCs from other types of muscle cells, characterized the phenotypical and transcriptomic changes in LMCs in aged vessels, and uncovered a pro-inflammatory microenvironment that suppresses the contractile apparatus in advanced-aged LMCs. Our findings provide a valuable resource to accelerate future research for the identification of potential drug targets on LMCs to preserve lymphatic vessel function as well as supporting studies to identify genetic causes of primary lymphedema currently with unknown molecular explanation.

SummaryTumor-draining lymph nodes are critical sites for generating tumor antigen-specific T cells and are associated with durable immune responses. However, lymph nodes are often the first site of metastasis and lymph node metastases portend worse outcomes. Through cross-species single cell gene expression analysis of breast cancer progression and metastasis to lymph nodes, we uncovered features that define the heterogeneity, plasticity, and immune evasion of cancer cells. Notably, a subpopulation of metastatic cancer cells in the lymph node were marked by high levels of MHC class II (MHC-II) gene expression both in mice and humans. Mechanistically, the IFN-γ and JAK/STAT signaling pathways mediate MHC-II expression in cancer cells. Ablation of IFNGR1/2 or CIITA, the transactivator of MHC-II, in cancer cells prevented tumor progression. Interestingly, MHC-II+ cancer cells lacked co-stimulatory molecule expression, engendered the expansion of regulatory T cells and blunted CD4+ effector T cells in the tumor-draining lymph nodes and favor tumor progression. Overall, our data suggests that cancer cell plasticity during breast cancer progression and metastasis to lymph nodes endows metastatic cells with the ability to avoid immune surveillance. These data provide the basis for new opportunities to therapeutically stimulate anti-cancer immune responses against local and systemic metastases.